The broad, long-term objective of this proposal is to elucidate the molecular mechanisms governing oxygen sensing, heme signaling and chaperone action in eukaryotes. Oxygen is vital for maintaining critical cellular functions in many living organisms, and heme is central to oxygen sensing and utilization. In humans, defects in oxygen sensing and regulation or in heme synthesis cause serious diseases in humans, including cancers, porphyrias, and respiratory and hematological diseases. Thus, understanding how oxygen is sensed and how heme and molecular chaperones promote oxygen and global gene regulation is important for improving human health. This proposal uses yeast as a model system for investigating oxygen sensing, heme signaling and chaperone action in eukaryotes. In yeast, heme mediates oxygen regulation of many genes by controlling the transcriptional activity of the heme activator protein Hapl. Molecular chaperones Hsp90 and Hsp70 bind to Hapl and promote heme regulation of Hapl activity. Experiments in this proposal will rigorously test the following hypotheses: (1) Hapl-multichaperone complexes bind directly to DNA and promote transcriptional aactivation and repression; (2) the oxygen level is sensed through heme synthesis; and (3) heme and Hapl play key roles in global oxygen sensing and global transcription regulation. The specific aims are (1) to clarify and compare the molecular mechanisms by which Hapl activates and represses transcription, (2) to dissect the molecular mechanism by which intracellular heme level is linked to oxygen level, and (3) to determine the global roles of heme and Hapl in oxygen sensing and regulation. Biochemical, genetic, and advanced microarray technologies and computational algorithms will be used to accomplish these aims. The gained knowledge should facilitate the understanding of the molecular mechanisms of oxygen sensing, heme signaling and chaperone action in higher eukaryotes.